MINISTRY OF EDUCATION AND TRAINING
DA NANG UNIVERSITY

Sesavanh MENVILAY

Research on Extraction and Determination
of the Chemical compositions from Rhizomes of three species
of Curcuma: Curcuma longa Linn., Curcuma aeruginosa Roxb. and
Curcuma mangga Valeton & Zijp. collected
in Champasack province, Laos

Major: Organic Chemistry
Code: 62440114

SUMMARY OF CHEMISTRY DOCTORAL THESIS

Da Nang – In 2019


The Thesis was completed in
DA NANG UNIVERSITY

Supervisors:

Prof. Dr. Dao Hung Cuong
Assoc. Prof. Dr. Le Tu Hai

Reviewer 1: ……………………………………………………….

Reviewer 2: ………………………………………………………


to solving the above problem, I chose the topic "Research on Extraction
and Determination of the Chemical compositions from the Rhizomes of
three species of Curcuma : Curcuma longa Linn., Curcuma aeruginosa
Roxb. and Curcuma mangga Valeton & Zijp. collected in Champasack
province, Laos " to carry out the contents of doctoral thesis.
2. Research purposes
- Development of the process of extraction, determination of chemical
composition of Curcuma in Laos by different methods; - To determine the


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curcumin content in some Curcuma in Laos; - Isolation, determination of
the formula of curcumin by spectral method.
3. Research object and scope
* Research object: Rhizome of Curcuma longa Linn., Rhizome of
Curcuma aeruginosa Roxb. and Rhizome of Curcuma mangga Valeton &
Zijp. collected in Champasack province, Laos.
* Research scope: Determine some physical and chemical properties of the
material such as moisture, ash content, composition and content of heavy
metals; Extract the essential oil of Rhizome of Curcuma longa Linn.,
Curcuma aeruginosa Roxb. and Curcuma mangga Valeton & Zijp. in Laos
by means of steam distillation; Determine the physical constants and
chemical indices of the essential oil of Curcuma longa Linn., Curcuma
aeruginosa Roxb. and Curcuma mangga Valeton & Zijp. in Laos; extract
the organic constituents in the dry rhizome of Curcuma longa Linn.,
Curcuma aeruginosa Roxb. and Curcuma mangga Valeton & Zijp. with
solvent of n- hexane, dichloromethane, ethyl acetate and methanol; extract
the curcumin in the dry rhizomes of Curcuma longa Linn. with KOH
solution; isolation, determine the chemical structure of curcumin from
coloring of Curcuma longa Linn.

layer

chromatography

and

column

chromatography. Solution spectrometry for determination of structure by
method: infrared spectrum (IR), (MS) mass spectrometry, one-way nuclear
magnetic resonance spectroscopy (1H. NMR,
1

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C-NRM, DEPT) and two-

1

way (HSQC, HMBC, H- H COSY).
5. New contributions of the thesis
According to lookup of references:
a. For the first time, the results of a relatively comprehensive study of
three species Curcuma (Curcuma longa Linn., Curcuma aeruginosa Roxb.
and Curcuma mangga Valeton & Zijp.) were announced. b. For the first
time, useful scientific information on the chemical composition of the
rhizomes of the Curcuma in Laos has been identified: 199 chemical
compounds have been identified in 3 kinds of Curcuma, of which Curcuma
longa Linn. is 97 compounds, Curcuma aeruginosa Roxb. is 111
compounds and Curcuma mangga Valeton & Zijp. is 117 compounds. c.

The thesis consists of 136 pages, consisting of parts:
Introduction: 4 pages
Chapter 1: Overview, 35 pages
Chapter 2: Materials and research methods, 13 pages
Chapter 3: Results and discussion, 69 pages.
Conclusion and Recommendations 15 pages


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CHAPTER 1. OVERVIEW
1. Introduction to the plant belonging to Curcuma genus, ginger family
1.1. Learn about the Curcuma genus, ginger family
Ginger family (zingiberaceae) is one of the relatively large plant
families, is a perennial herb family with horizontal rhizomes or creation of
tuber, in which many species are decorative plants, herbs, or medicinal
plants. The most important members of this family are ginger, Curcuma,
galanga, Myristica fragrans and Amomum villosum. The ginger family
includes 47 genera and about 1300 species, in Vietnam and Indochinese
countries, Curcuma genus includes 19 species, in Bangladesh there are 1620 species, in India, China and Indochina, there are 20-25 species, in
Malaysia, there are 16-20 species, in Nepal, there are 10-15 species, in
Philippines, there are 12-15 species, in Thailand, there are 30-40 species.
Therefore, there is generally no consensus among the literature on the
number of species in the Curcuma genus.
1.2. Plant characteristics, distribution and chemical composition of some
Curcuma species
Turmeric (Curcuma) belonging to Ginger family (zingiberaceae) is not
only a perennial herb, it can reach a height of more than 1 meter. The plant
creates branch highly, with cylindrical form, strong rhizome, fleshy and has
many branches, rhizome, fragrant smell, grows into tuber. Leaves are flat
with spearhead or oval shape, grow with flowers or grow after flowers, the

diseases such as stomach pain, arthritis, wounds, abdominal pain, cough,
irregular menstruation, tumor treatment, liver detoxification....
1.5. Research history on Curcuma
- Daube (1870) obtained crystalline curcumin and was identified as 1.6
heptadiene -3.5- dione- 1.7-bis (4-hydroxyl-3-methycylphenyl) - (1E, 6E)
or diferuloylmethane. Curcumin's diferuloylmethane frame is confirmed.
- According to Mr. Phan Tong Son et al. (1998), in Vietnam the main
components of the rhizome's essential oil of Curcuma aromatica Salisb.
are: 1.8 cineole; tecpinolene;

- elemene; humulene;

– elemene,


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especially curzerenone (37.78%) and germa-1 (10), 4.7 (11) - triene-8-on
(11.22%).
1.6. Research history on the structure of curcumin
- In 1953, Srinivasan K.R demonstrated by chromatography on a silic
column that curcumin is a mixture, of which curcumin I accounts for 60%,
curcumin II accounts for 24% and curcumin III accounts for 14%.
1.6.1. Structure of Curcumin


In nature curcumin exists in four forms as:
- Curcumin I: Molecular formula: C21H20O6

Structural formula:


1.6.3. Chemical properties of curcumin
1.6.4. Biological activities
1.7. Curcumin application
1.7.1. In the medical profession
1.7.2. In industry
1.7.3. Some folk remedies used in life
1.7.4. Nano curcumin
CHAPTER 2 : MATERIALS AND RESEARCH METHODS
2.1. Materials
2.2. Research methods
2.2.1. Mass analysis method
a. Determination of moisture content of raw materials; b. Determination
of ash content


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2.2.2. Atomic absorption spectroscopy method AAS
2.2.3. Identification Method of Chemical Composition GC-MS
2.2.4. Methods of researching essential oil
a. Extraction of essential oil; b. Experimental collection of essential oil;
c. Determination of physical and chemical properties of essential oil; d.
Identification of chemical composition in essential oil.
2.2.5. Research methods of organic extract
a. Extraction for collection of organic extract; b. Extraction for collection
of extract; c. Identification of chemical composition in the extract.
2.2.6. Curcumin extraction with KOH solution
2.2.7. Isolation to determine the formula of curcumin
CHAPTER 3: RESULTS AND DISCUSSION
3.1. Survey results of some physico-chemical indicators
3.1.1. Determine the moisture, ash content of the material

3.2. Research results of essential oil of Curcuma in Laos
3.2.1. Steam distillation
Results obtained: average essential oil content of Curcuma longa Linn. is
0,237%; Curcuma aeruginosa Roxb. is 0,355% and Curcuma mangga
Valeton & Zijp. is 0,235%.
3.2.2. Sensory evaluation of rhizome essential oil of Curcuma in Laos
Essential oil of Curcuma longa Linn. has pale yellow, characteristic
aroma and spicy taste. Essential oil of Curcuma aeruginosa Roxb. has pale
black-yellow, characteristic aroma, spicy and bitter taste. Essential oil of
Curcuma mangga Valeton & Zijp. has pale yellow, characteristic aroma
and pungent taste.

Fig. 3.1. Essential oil of Curcuma in Laos
3.2.3. Results of the determination of physicochemical parameters of
rhizome's essential oil of Curcuma longa Linn., Curcuma aeruginosa
Roxb. and Curcuma mangga Valeton & Zijp. in Laos.


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- Proportion result of essential oil of Curcuma longa Linn. is 0,965,
Curcuma aeruginosa Roxb. is 0,963 and Curcuma mangga Valeton & Zijp.
is 0,966. This value is equivalent to turmeric essential oil in the world and
predictions of the chemical composition of essential oil are mainly
hydrocarbons and alcohol. - The results of the refractive index obtained
through the measurement of rhizome's essential oil of Curcuma longa
Linn.1,511, Curcuma aeruginosa Roxb.1,512 and Curcuma mangga
Valeton & Zijp. 1,472. This index corresponds to the average value of
refractive index reported by many publications (1,555-1,520). - Result of
determination of acid index of rhizome's essential oil of Curcuma longa
Linn. 2,60, Curcuma aeruginosa Roxb. 2,42 and Curcuma mangga Valeton

The identification results of the chemical composition of essential oil of
Curcuma mangga Valeton & Zijp. in Laos by GC-MS have identified 30
constituents. The constituent with highest percentage is zingiberene
(10,72%); followed by

– mycrene (10,70%); eucalyptol (9,71%). The

remaining constituents accounts for 0,03% -3,34%. - The summary result
of the chemical compositions identified in the essential oil of Curcuma in
Laos has 42 constituents, including 30 constituents in the essential oil of
Curcuma longa Linn.; 25 constituents in the essential oil of Curcuma
aeruginosa Roxb.; 30 constituents in the essential oil of Curcuma mangga
Valeton & Zijp. There are 16 constituents available in all three types of
turmeric essential oil. Among the identified ingredients, we mention
compounds with strong biological activity such as ar-tumerone,
caryophyllene, eucalyptol, curzerene.
3.3.4. Comparing the chemical composition of Lao turmeric essential
oil with the same turmeric essential oil in cuontries
- There are 30 constituents in Champasack essential oil of Curcuma longa
Linn. have identified, higher than Kon Tum essential oil of Curcuma longa
Linn. with 10 constituents. There are 9 constituents available in all two
types of turmeric essential oil. The constituent with highest percentage is
zingiberene (22,98%), higher than 10 times compared with percentage of
zingiberene (7,16%) in Kon Tum essential oil of Curcuma longa Linn.


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- There are 25 constituents in the Champasack essential oil of Curcuma
aeruginosa Roxb. have identified, higher than Huong Hoa -Quang tri
essential oil of Curcuma aeruginosa Roxb. with 14 constituents; 9

a. Effect of time to effective collection of extracts : The results obtained
for the appropriate extraction time were 10 hours with a percentage of
extracted volume of 4,100%.
b. Chemical composition of Curcuma longa Linn. in n-hexane extract
The identification results of the chemical composition of hexane extract
from Curcuma longa Linn. by GC-MS, have identified 14 constituents. The
constituent with highest percentage is ar-tumerone (22,65%). The
remaining constituents accounts for 6,66% - 0,10%.
3.4.2. Chemical composition of n-hexane extract from Curcuma
aeruginosa Roxb.
a. Effect of time to effective collection of extracts: The results obtained
for the appropriate extraction time were 10 hours with a percentage of
extracted volume of 10,333%.
b. Chemical composition of Curcuma aeruginosa Roxb. in n-hexane
extract : The identification results of the chemical composition of n-hexane
extract from Curcuma aeruginosa Roxb. by GC-MS, have identified 24
constituents. The constituent with highest content was curzerene with
(44,02%), followed by -elemene with (15,47%), the remaining constituent
accounts for 0,01% -3,85%.
3.4.3. Chemical composition of n-hexane extract from Curcuma
mangga Valeton & Zijp.
a. Effect of time to effective collection of extracts : The results obtained
for the appropriate extraction time were 8 hours with a percentage of
extracted volume of 6,709%.
b. Chemical composition of Curcuma mangga Valeton & Zijp in nhexane extract : The identification results of the chemical composition of


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n-hexane extract from Curcuma mangga Valeton & Zijp. by GC-MS, have
identified 27 constituents. The constituent with highest content was

for the appropriate extraction time were 10 hours with a percentage of
extracted volume of 5,717%.


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b.

Chemical

composition

of

Curcuma

aeruginosa

Roxb.

in

dichloromethane extract : The identification results of the chemical
composition of dichloromethane extract from Curcuma aeruginosa Roxb.
in Laos by GC-MS, have identified 22 constituents. The constituent with
highest content was curzerene with (42,26%), followed by -elemene with
(15,37%), the remaining constituent accounts for 0,01% 3,57%.
3.5.3. Chemical composition of dichloromethane extract from Curcuma
mangga Valeton & Zijp.
a. Effect of time to effective collection of extracts: The results obtained for
the appropriate extraction time were 8 hours with a percentage of extracted

extract by GC-MS, have identified 16 constituents. The constituent with
highest content was ar-tumerone (17,19%), the remaining constituent
accounts for 7,30% - 0,10%.
3.6.2. Chemical composition of ethyl acetate extract from Curcuma
aeruginosa Roxb.
a. Effect of time to effective collection of extracts : The results obtained
for the appropriate extraction time were 8 hours with a percentage of
extracted volume of 4,545%.
b. Chemical composition of Curcuma aeruginosa Roxb. in ethyl acetate
extract : The identification results of the chemical composition of ethyl
acetate extract from Curcuma aeruginosa Roxb. in Laos by GC-MS, have
identified 20 constituents. The constituent with highest content was
curzerene with (36,72%), followed by

-elemene with (13,18%), the

remaining constituent accounts for 0,02% - 2,34%.
3.6.3. Chemical composition of ethyl acetate extract from Curcuma
mangga Valeton & Zijp.
a. Effect of time to effective collection of extracts : The results obtained
for the appropriate extraction time were 8 hours with a percentage of
extracted volume of 5,334%.
b. Chemical composition of Curcuma mangga Valeton & Zijp. in ethyl
acetate extract : The identification results of the chemical composition of
ethyl acetate extract from Curcuma mangga Valeton & Zijp. in Laos by
GC-MS, have identified 15 constituents. The constituent with highest
content was -elemenone with (7,16%), the remaining constituent accounts


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have identified 19 constituents. The constituent with highest content was
curzerene with (41,17%), followed by

-elemene with (14,03%), the

remaining constituent accounts for 0,03% - 2,29%.
3.7.3. Chemical composition of methanol extract from Curcuma
mangga Valeton & Zijp.
a. Effect of time to effective collection of extracts : The results obtained
for the appropriate extraction time were 8 hours with a percentage of
extracted volume of 7,927%.
b. Chemical composition of Curcuma mangga Valeton & Zijp. in
methanol extract : The identification results of the chemical composition
of methanol extract from Curcuma mangga Valeton & Zijp. in Laos by
GC-MS, have identified 21 constituents. The constituent with highest
content was curzerene with (19,63%), followed by

-elemene with

(6,04%), the remaining constituent accounts for 0,04% -3,14%.

- The

summary result of the chemical compositions identified in the methanol
extract of Curcuma in Laos has 35 constituents, including 15 constituents
in the extract of Curcuma longa Linn.; 19 constituents in the extract of
Curcuma aeruginosa Roxb.; 21 constituents in the extract of Curcuma
mangga Valeton & Zijp. There are 3 constituents available in all three


(A)

(B)

Fig. 3.4. UV-VIS spectra of colorants (A) and standard curcumin (B)


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The results determine that the curcumin content contained in the colorant
crystallined in HPLC table are shown in Figure 3.5

Fig. 3.5. HPLC chromatography for curcumin quantitative
The results showed that the curcumin content in the sample was 90,899%.
3.9.3. Isolatation and determination of the Curcumin formulation
Column chromatography using silicagel 60 F524 of Merck brand. The
appropriate solvent system for the isolation of the substance is the n-hexane
system: ethyl acetate with the rate ranging from (65:35) to (15:85). The
collection phase is from containers no. 35 to 82 for a rounded
chromatogram with a dark yellow color, symbol: M1 with Rf = 0.625. - MS
Mass spectrometry of M1 substance for molecular pic ion [M-H] has the
mass number m/z = 337, the molecular mass of M1 is 338. - IR spectrum of
M1 substance gives the characteristic signal at v (cm1): 3308; 1574; 1510;
1436; 1271; 1139; 967; 824. - 1H-NMR spectrum of M1 substance showed
the signal of 18 protons, 13C-NMR spectrum showed that the signal of 20 C
atoms combined with MS spectra allowed to predict molecular formula of
M1 substance as C20H18O5. Symmetric signals on 1H-NMR spectrum and
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C-NMR with the number of protons and C in M1 corresponds fully to the

Linn.: 2,60, Curcuma aeruginosa Roxb.: 2,42 and Curcuma mangga
Valeton & Zijp.: 2,39); Ester index (Curcuma longa Linn.: 21,75, Curcuma
aeruginosa Roxb.: 26,61 and Curcuma mangga Valeton & Zijp.: 18,52);


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Saponification index (Curcuma longa Linn.: 32,87, Curcuma aeruginosa
Roxb.: 28,99 and Curcuma mangga Valeton & Zijp.: 20,92).
3. By gas chromatography mass spectrometry (GC-MS) have been
identified in Champasck essential oil of Curcuma in Laos, of which 30
constituents are found in essential oil of Curcuma longa Linn.; 25
constituents in essential oil of Curcuma aeruginosa Roxb. and 30
constituents in essential oil of Curcuma mangga Valeton & Zijp. There are
16 constituents available in all three types of turmeric essential oil. Thereby
comparing the chemical composition of Champasack essential oil of
Curcuma in Laos with other countries, the percentage of constituents in
general is not much different only varies the number of constituents
4. By the Soxhlet method was determined the appropriate extraction time
to obtain the highest n-hexane and dichloromethane extract for each of
rhizome of Curmuma in Laos: Curcuma longa Linn.: 10 hours; Curcuma
aeruginosa Roxb.: 10 hours and Curcuma mangga Valeton & Zijp.: 8
hours and identified constituents in the n-hexane extraction, of which 14
constituents in extract of Curcuma longa Linn., 24 constituents in extract of
Curcuma aeruginosa Roxb., 27 constituents in extract of Curcuma mangga
Valeton & Zijp.There are 4 constituents available in all three types of
turmeric extract. and identified constituents in the dichloromethane
extraction, of which 22 constituents in extract of Curcuma aeruginosa
Roxb., 23 constituents in extract of Curcuma mangga Valeton & Zijp.
There are 3 constituents available in all three types of turmeric extract.
- Determined the appropriate extraction time to obtain the highest amount